In recent years, the Maillard reaction between proteins and reducing sugars such as glucose and the like is drawing attention as a cause of degenerating proteins or as a cause of various complications of diabetes.
The Maillard reaction is a reaction in which a mixture of an amino acid or a protein together with a reducing sugar turns into brownish color upon heating. In a first stage, an amino group of a protein or an amino acid reacts with an aldehyde group of a sugar to form a Schiff base compound. A 3-deoxyglucosone derivative is an intermediate metabolite formed as a result of the Maillard reaction and exhibits a strong crosslinking action to proteins.
The crosslinking, polymerization and degeneration of proteins are greatly affected by the Maillard reaction, even for those proteins having particularly long life (crystalline, collagen) suggesting that it is closely related to the aging and degeneration of proteins.
It has therefore been attempted to determine the 3-deoxyglucosone derivatives. In practice, however, there is available no suitable internal standard substance, and correct determination has not been carried out. That is, according to a conventional absolute method, the 3-deoxyglucosone derivative has been directly determined, or a reduced product thereof obtained with NaBD.sub.4 has been utilized (e.g., K. Knecht, et al., Archives Biochem Biophy., 294, 130, (1992)).
The 3-deoxyglucosone derivatives are intermediate metabolites of the Maillard reaction and they could not be highly reliably analyzed from body materials and fluids such as blood, urine, hair, skin and tear or from the living body tissues such as various organs, despite attention having been given to such derivatives in regard to aging and various disease conditions such as complications of diabetes.
In recent years, a method has been proposed for determining the 3-deoxyglucosone derivatives owing to the progress in view of the high sensitivity and easy operation in gas chromatography/mass spectrometry which relies on gas chromatography using a mass spectrometer as a detector.
According to the analysis disclosed in Knecht, et al., a 3-deoxyglucosone derivative is reduced with NaBD.sub.4 or NaBT.sub.4 to form a 3-deoxyhexitol, followed by conversion into an acylated derivative thereof which is subjected to the gas chromatograph mass spectrometer.
According to this analytical method, either a 3-[1,2-.sup.2 H]-deoxyhexitol or a 3-[1,2-.sup.3 H]-deoxyhexitol, which is an isotope-labelled compound wherein first and second positions are substituted with deuterium, is used as an internal standard. However, this isotope-labelled compound has a mass number which is larger by only two than a naturally existing one because it is a compound obtained by reducing the 3-deoxyglucosone derivative, and has poor stability in an aqueous solution.
Being added in the stage of preparing samples, the isotope-labelled compound serves as an indication of recovery rate and conversion rate. In order that the determination can be carried out maintaining high precision, it is desired that the isotope-labelled compounds that serve as an internal standard have a different mass number yet also have the same chemical properties as a naturally existing compound and a label at a stable position in the molecules. Therefore, such a substance has been sought.